Contrast agents

ABSTRACT

The invention relates to ultrasound contrast agents comprising vesicles comprising a protein capable of formation of gas-containing vesicles, wherein the vesicles contain gas which comprises sulphur hexafluoride or a low molecular weight fluorinated hydrocarbon. These contrast agents exhibit stability in vivo upon administration so as to permit ultrasound visualization while allowing rapid subsequent elimination from the system.

This invention relates to novel contrast agents, more particularly tonew gas-containing or gas-generating contrast agents of use indiagnostic ultrasonic imaging.

It is well known that ultrasonic imaging comprises a potentiallyvaluable diagnostic tool, for example, in studies of the vascularsystem, particularly in cardiography, and of tissue microvasculature. Avariety of contrast agents has been proposed to enhance the acousticimages so obtained, including suspensions of solid particles, emulsifiedliquid droplets, gas bubbles and encapsulated gases or liquids. It isgenerally accepted that low density contrast agents which are easilycompressible are particularly efficient in terms of the acousticbackscatter they generate, and considerable interest has therefore beenshown in the preparation of gas-containing and gas-generating systems.

Initial studies involving free gas bubbles generated in vivo byintracardiac injection of physiologically acceptable substances havedemonstrated the potential efficiency of such bubbles as contrast agentsin echocardiography; such techniques are severely limited in practice,however, by the short lifetime of the free bubbles. Interest hasaccordingly been shown in methods of stabilising gas bubbles forechocardiography and other ultrasonic studies, for example usingemulsifiers, oils, thickeners or sugars.

WO 80/02365 discloses the use of gelatin encapsulated gas microbubblesfor enhancing ultrasonic images. Such microbubbles do not, however,exhibit adequate stability at the dimensions preferred for use inechocardiography (1-10 μm) in view of the extreme thinness of theencapsulating coating.

EP-A-0327490 discloses, inter alia, ultrasonic contrast agentscomprising a microparticulate synthetic biodegradable polymer (e.g. apolyester of a hydroxy carbonic acid, a polyalkyl cyanoacrylate, apolyamino acid, a polyamide, a polyacrylated saccharine or apolyorthoester) containing a gas or volatile fluid (i.e. having aboiling point below 60° C.) in free or bonded form. Emulsifiers may beemployed as stabilisers in the preparation of such agents, but suchemulsifiers do not chemically interact with the polymer.

U.S. Pat. No. 4,774,958 discloses the use of microbubble dispersionsstabilised by encapsulation in denatured protein, e.g. human serumalbumin (HSA). Such systems permit the production of microbubble systemshaving a size of e.g. 2-5 μm but still do not permit efficientvisualisation of the left heart and myocardium.

Other ultrasound contrast agents using proteins as encapsulating agentshave been described in the literature, for example in EP 0359 246(Molecular Biosystems), U.S. Pat. No. 4,832,941 (Max-PlanckGessellschaft), U.S. Pat. No. 4,844,882 (Molecular Biosystems), WO84/02838 (Feinstein), U.S. Pat. No. 4,572,203 (Feinstein), EP 0077 752(Schering), U.S. Pat. No. 4,747,610 (The Regents of the University ofCalifornia), WO 80/02365 (Rasor), U.S. Pat. No. 4,774,958 (Feinstein),U.S. Pat. No. 4,718,433 (Feinstein), EP 0224 934 (Feinstein).

The only protein-based ultrasound contrast agent under commercialdevelopment consists of a suspension of gas-filled albumin, Albunex®,prepared by sonication of a solution of albumin.

Albumin based ultrasound contrast agents are described in the followingpublications:

Feinstein et al. in Circulation 78S, 565 (1988), Reisner et al. inCirculation 78S, 565 (1988), Dick et al. in Circulation 78S, 565 (1988),Armstrong et al. in Circulation 78S, 565 (1988), Desir et al. inCirculation 78S, 566 (1988), Heidenreich et al. in Circulation 78S, 566(1988), Keller et al. in Circulation 78S, 567 (1988), Barnhart et al. inContrast Media Research (1989), Silverman et al. in Circulation 80S, 369(1989), Silverman et al. in Circulation 80S, 349 (1989), Segar at al. inClin.Res. 37, 294 (1989), Heidenreich et al. in Circulation 80S, 370(1989), Reiser et al. in Circulation 80S, 370 (1989), Heidenreich et al.in Circulation 80S, 566 (1989), Shandas et al. in Circulation 82, 95(1990), Geny et al. in Circulation 82, 95 (1990), Ten-Cate et al. in EurHeart J. 19, 389 (1989), Feinstein et al. in Echocardiography 6, 27(1989), Zotz et al. in Eur Heart J. 11, 261 (1990), Ten-cate et al. inEur Heart J. 11, 261 (1990), Barnhart et al. in Invest Radiol 25S, 162(1990), Keller et al. in J. Am Soc Echo 2, 48 (1989), Bleeker et al. inJ. Acoust Soc Am 87, 1792 (1990), Feinstein et al. in J. Am. Coll.Cardiol 16, 316 (1990), Kaul et al. in J. Am Coll. Cardiol 15, 195(1990), Bleeker et al in J. Ultrasound Med 9, 461 (1990), Hilpert et al.in Radiology 173, 361 (1989), and Shapiro et al. in J. Am. Coll. 16,1603 (1990).

However, as indicated above, ultrasound contrast agents based ongas-filled protein microspheres are unstable in vivo, and there is roomfor improvement of such products. Segar et al. have, in Advances inEchocardiography (Sep. 21-22-1989), concluded that batch, mixingpressure, mixing time and medium all affect the left atrium contrastwith such protein based products.

Feinstein et al. have in J. Am. Coll. Cardiol 16, 316 (1990) publishedthat irrespective of dose group, a cavity opacification with albuminmicrospheres was seen in the right ventricle in 88% of the injectionsand in the left ventricle in 63% of the injections. Shandas et al. havein Circulation 82, 95 (1990) raised questions about the pressure relatedstability of gas filled albumin microspheres and Shapiro et al. haverecently published in J. Am. Coll. Cardiol 16, 1603 (1990) lack ofultrasound myocardial contrast enhancement after administration ofsonicated albumin.

Feinstein has in EP 0224 934 on page 4,8 and claim 9, U.S. Pat. No.4,718,433 columns 3 and 5 and U.S. Pat. No. 4,774,958 columns 3 and 5suggested chemical denaturation to stabilize albumin gas bubbles:

-   -   “The microbubbles formed from 5% albumin may, in the        alternative, be stabilized to form a commercially, clinically        usable contrast agent by treatment with various chemical agents        which chemically denature, or “fix”, the protein, and        derivatives thereof. Chemical denaturation of the protein (or        derivatives) may be accomplished by either binding the protein        with a protein-reactive aldehyde, such as glutaraldehyde. For        the latter procedure of stabilizing the invented microbubble        contrast agent, the microbubbles may be reacted with 0.25 grams        of 50% aqueous glutaraldehyde per gram of protein at pH 4.5 for        6 hours. The treated contrast agent is then gently and        extensively washed to remove as much of the unreacted        glutaraldehyde as possible.”

Various denaturing chemicals or cross linking agents for proteins havebeen described in the literature. (See for example Methods Enzymol 172,584 (1989) and Chemical Reagents for Protein Modification, Volume II,page 123, CRC Press Inc.)

However it is important that any contrast agent should be rapidlyeliminated from the subject in a short term after use, e.g. preferablyhaving a half life of not more than 48 hours. Crosslinking byglutaraldehyde or formaldehyde may not always be effective in providingan adequate balance between stability during ultrasound visualisationand rapid elimination. The protein itself, being human serum albumin, isnot rapidly degraded by vascular enzymes and reagents such asglutaraldehyde do not form readily biodegradable bonds with the protein.

The present invention is based on the concept of crosslinking theprotein shells of microbubbles to introduce biodegradable linkinggroups, thus providing ultrasound contrast agents with adequatestability for the duration of ultrasound visualisation but sufficientbiodegradability to permit rapid elimination subsequently.

According to the present invention, therefore, we provide ultrasoundcontrast agents comprising microbubbles of gas or a gas precursorencapsulated in a shell of protein crosslinked with biodegradablecrosslinking groupings.

Biodegradable linkages which may be used include amide, imide, imine,ester, anhydride, acetal, carbamate, carbonate, carbonate ester anddisulphide groups. At least one such group should preferably be presentin the crosslinking grouping. In general, any esters will bebiodegradable particularly those containing the grouping —CO.O— or—O.CO.O—. One particularly useful class of biodegradable ester groupingshas the structure—(Y)_(n).CO.O.C(R¹R²).O.CO.(Z)_(n)—(where Y and Z, which may be the same or different, are —O—, —S— or—NR³—; the symbols n, which may be the same or different, are zero or 1;R¹ and R², which may be the same or different, are hydrogen atoms orcarbon-attached monovalent groups or together represent acarbon-attached divalent organic group; and R³ is a hydrogen atom or anorganic group. Y and Z are preferably —O—. Such groups generally degradeto eliminate a compound R¹R²CO and either form carboxyl groups on theresidue or, in the case of carbonate esters, may eliminate carbondioxide to form hydroxyl groups on the residue.

R¹, R² and R³ may each be a hydrocarbyl or heterocyclic group, forexample having 1-20 carbon atoms, e.g. an alkyl or alkenyl group(preferably having up to 10 carbon atoms), a cycloalkyl group(preferably having up to 10 carbon atoms), an aralkyl group (preferablyhaving up to 20 carbon atoms), an acyl group (preferably having up to 20carbon atoms) or a heterocyclic group having up to 20 carbon atoms andone or more heteroatoms selected from O, S and N; such a hydrocarbyl orheterocyclic grouping may carry one or more functional groups such ashalogen atoms or groups of the formulae —NR⁴R⁵, —CONR⁴R⁵, —OR⁶, —SR⁶ and—COOR⁷, where R⁴ and R⁵, which may be the same or different, arehydrogen atoms, acyl groups or hydrocarbyl groups as defined for R¹ andR²; R⁶ is a hydrogen atom or an acyl group or a group as defined for R¹or R² and R⁷ is a hydrogen atom or a group as defined for R¹ or R²;where R¹ and R² represent a divalent grouping, this may for example bean alkylene or alkenylene group (preferably having up to 10 carbonatoms) which may carry one or more functional groups as defined above.In general R¹ and R² are preferably hydrogen or small groups such asC₁₋₄ alkyl groups.

The protein component can be any protein or derivative thereof includingpolyamino acids. Albumin, gelatin and γ-globulin are representativecompounds. The protein, for instance albumin, can be obtained frombiological sources, for example from human or animal blood, or producedby a lower organism using recombinant technology. A typical method forpreparation of human serum albumin by fermentation is described in WO9002808 (Delta Biotechnology Ltd.).

According to a further feature of the invention, we provide a processfor the preparation of microbubble ultrasound contrast agents in which agas or a gas precursor is encapsulated in a protein which is crosslinkedwith biodegradable crosslinking groups.

The crosslinking of the protein can be effected before, during or afterencapsulation. It is preferred to encapsulate, e.g. by formingmicrobubbles, first and to effect crosslinking subsequently.

The crosslinking agent may be a compound of the formula (I)A¹-X-A²   (I)where X is a linking group containing one or more biodegradable linkagesand the groups A¹ and A², which may be the same or different, arefunctional groups reactive with proteins.

The group X may carry further groups reactive with proteins to providean even greater degree of crosslinking.

Preferably, the group X should have a chain length of not more than 30atoms.

The group X may thus be of the form—R⁸-E-R⁹—where R⁸ and R⁹, which may be the same or different, are divalentorganic groups, for example alkylene or alkylidene groups having 1-12carbon atoms, which may carry groups reactive with proteins and/orfurther inert groups, and the group E is an ester grouping, for exampleof the formula —O.CO—, —O.CO.O— or —(Y)_(n).CO.O.C(R¹R²).O.CO.(Z)_(n)-as defined above.

Crosslinking agents of the formulaA¹.R⁸.(Y)_(n).CO.O.C(R¹R²).O.CO.(Z)_(n).R⁹.A²where A¹, A², R¹, R², R⁸, R⁹, n, Y and Z have the above meanings may beprepared by reaction of an acid of the formula A¹.R⁸.(Y)_(n).CO.OH or aform thereof in which A¹ and any other reactive groups are protected (ora functional derivative thereof) with a compound of the formulaL¹.C(R¹R²).L² where L¹ is a leaving group such as a halogen atom ormesyloxy or tosyloxy and L² is a group as defined for L¹ (giving asymmetrical di-ester) or a group of the formula —O.CO.(Z)_(n).R⁹.A² or aprotected form thereof, if necessary followed by deprotection. Thefunctional derivative of the acid may for example be a salt, e.g. thepotassium salt. The reaction will normally be carried out in solution,for example in a polar solvent such as dimethylformamide. Protectinggroups for A¹ and A² may be those conventional in the art. Preferredprotecting groups for aldehydes include acetals, e.g. cyclic acetalssuch as dioxolan.

The compound L¹.C(R¹R²).O.Co.(Z)_(n).R⁹. A², where L¹ is halogen, may beprepared from R¹R².CO by reaction with a compound of the formulaHal.CO.(Z)_(n).R⁹.A² (where Hal represents a halogen atom) in thepresence of a base such as pyridine.

Apart from aldehyde groups, which are preferred, the groups A¹ and A²may be activated carboxyl groups, such as N-hydroxysuccinimidyl groups(especially water solubility-enhanced sulphonated N-hydroxysuccinimidylderivatives), imidoesters, halo-nitroaryl groups, nitrene precursorgroups such as azidophenyl, carbene precursor groups, ketone groups,isothiocyanate groups etc.

Any biocompatible gas maybe employed in the contrast agents of theinvention, for example air, nitrogen, oxygen, hydrogen, nitrous oxide,carbon dioxide, helium, argon, sulphur hexafluoride and low molecularweight optionally fluorinated hydrocarbons such as methane, acetylene orcarbon tetrafluoride. The gas maybe free within the microbubble or maybe trapped or entrained within a containing substance. The term “gas” asused herein includes any substance in the gaseous form at 37° C.

Gas precursors include carbonates and bicarbonates, e.g. sodium orammonium bicarbonate and aminomalonate esters.

For applications in echocardiography, in order to permit free passagethrough the pulmonary system and to achieve resonance with the preferredimaging frequency of about 0.1-15 MHz, it may be convenient to employmicrobubbles having an average size of 0.1-10 μm, e.g. 1-7 μm.Substantially larger bubbles, e.g. with average sizes of up to 500 μm,may however be useful in other applications, for examplegastrointestinal imaging or investigations of the uterus or Fallopiantubes.

As indicated above the microbubbles may be stabilised by incorporationof particulate material together with the encapsulated gas. Suchparticles include, for example, silica and iron oxide. The preferredparticle size for such stabilising particles is in the range 1 to 500nm, depending on the size of the microbubbles. The particles should besuch that they are only partially wetted by the fluid medium used todisperse the micelles, i.e. the contact angle between the material ofthe particles and the fluid should be about 90 degrees.

The stabilising particles may carry functional groups which willinteract with the protein to form covalent or other linkages. Colloidalsilica particles may have a particle size in the range 5-50 nm and maycarry silanol groups on the surface which are capable of interactionwith the protein by hydrogen bonding or by forming covalent bond.

The protein may stabilize the gas or gas precursor by forming amonolayer at the interface between the liquid medium and the gas or gasprecursor system, or by forming vesicles consisting of one or morebilayers containing the gas or gas precursor.

The stabilisation of the system by monolayers or the formation of thevesicles may be activated, as fully described in the literature, bysonication or even shaking of the protein material mixture in theappropriate medium, or the vesicles may be formed by any conventionalliposome/vesicle-forming principle.

The stabilized microbubbles may be dried or freeze-dried or thenon-aqueous phase may be evaporated. The resulting dried system may beresuspended in any physiological acceptable solvent such a saline orphosphate buffer, optionally using a suspending or emulsifying agent.

A gas entrapped system may be obtained by using a gas precursor or thegas itself may be entrapped. The gas may be entrapped into theamphiphile mixture simply by vigorously shaking the mixture in thepresence of air,. i.e. creating a gas-in-liquid emulsion as described inU.S. Pat. No. 4,684,479. Another well established method, described i.e.in U.S. Pat. No. 4,774,958 for creating a gas-containing bubble is bysonication of the mixture in the presence of air. Another well knownmethod is passing the gas through a syringe into the mixture of theprotein and the liquid. As described in U.S. Pat. No. 3,900,420 themicrogas-emulsion may be created by using an apparatus for introducinggas rapidly into a fast-flowing liquid. A region of low pressure iscreated in a liquid containing the protein material. The gas is thenintroduced to the region of low pressure and the gas-in-liquid system isobtained by pumping the liquid through the system.

By using the principle of electrolysis it is possible to generate thegas to be entrapped directly in a container containing the proteinmaterial. The electrolytes necessary for the electrolysis may even helpto further stabilize the protein material. An aqueous solutioncontaining electrolytes may generate hydrogen gas at the cathode andoxygen at the anode. The electrodes may be separated by a salt bridge.On adding hydrazine nitrogen gas may be generated at the anode. Usingthe Kolbe reaction, one may also generate CO₂ from carboxylic acidsusing electrolysis.

As described above, microbubbles may be obtained by forming liposomes orvesicles consisting of one or more bilayers. These vesicles may beformed at elevated pressure conditions in such a way that the gas isentrapped in the vesicles.

In one procedure according to the invention, encapsulation is effectedby agitation or sonication of the protein in an aqueous medium to yielda protein foam which is dried and thereafter suspended in a solution ofthe crosslinking agent in a polar organic solvent (e.g. a sulphoxidesuch as dimethyl sulphoxide) which is capable of wetting the proteinfoam.

The following Examples are given by way of illustration only:

Preparation 1 Methylene bis(α-formylacetate)

The preparation of the starting material, the dioxolan-protectedaldehyde methyl α-formylacetate, is described by T. Hosokawa et al. J.Org. Chem. Soc. 52, (1987) 1758-1764. The protected aldehyde (6.0 g,3.75 mmol) is treated with a mixture of 2N aqueous potassium hydroxideand tetrahydrofuran 20:80 (v/v) at reflux for 8 hours. The pH isadjusted to 8 using diluted HCl, and the mixture is evaporated todryness. The solid is mixed with 100 ml freshly distilled and drieddimethylformamide, and after 30 minutes at 60° C. the undissolvedmaterial is filtered off. Diiodomethane (150 μl, 1.87 mmol) is addeddropwise during 5 minutes to the solution at 60° C. as described in WO89/00988 page 13 (NYCOMED AS). The precipitate is removed by filtrationafter stirring for 4 days, and the solvent removed at reduced pressure.The dioxolan protection is removed as described by P. A. Grieco et al.J. Am. Chem. Soc. 99, (1977) 5773-5780—the residue is dissolved intetrahydrofuran (60 ml), 5% aqueous HCl (20 ml) is added and the mixtureis stirred for 20 hours at ambient temperature. The reaction mixture isevaporated to dryness under reduced pressure to yield the titlecompound.

Preparation 2 Methylene dimethacrylate

A solution of potassium hydroxide (1.00 M, 40.00 ml) is added tomethacrylic acid (3.44 g, 40.00 mmol) at 0° C. and the solution freezedried for 16 hours. Dry dimethylformamide (230 ml) is added and thesuspension heated to 60° C. under a dry nitrogen atmosphere.Diiodomethane (1.61 ml, 20.00 mmol) is added in two portions during 10min. and the reaction mixture left for 4 days at 60° C. The solvent isremoved under reduced pressure (0.05 mm Hg), before diethyl ether (140ml), saturated aqueous sodium hydrogen carbonate (50 ml) and water (50ml) are added. The aqueous layer is extracted with diethyl ether (6×60ml) and the combined ether extracts washed with water (4×50 ml), dried(MgSO₄), and evaporated to give 2.63 g (72%) of the title compound. ¹HNMR (60 MHz, CDCl₃): δ 1.97 (2×CH₃, m), 5.63 (2×H—C═, m), 5.88 (CH₂, s),6.18 (2×H—C═, m). IR (film, c=⁻¹): 2987 (w), 2962 (w), 2930 (w), 1732(str), 1638 (w), 1454 (w), 1315 (w), 1295 (w), 1158 (w), 1100 (str),1012 (m), 989 (m). This product may be used in accordance with theinvention, for example to crosslink acrylamide polymers.

Preparation 3 Methylene diacrylate

A solution of potassium hydroxide (1.00 M, 40.00 ml) is added to acrylicacid (2.88 g, 40.00 mmol) at 0° C. and the solution freeze dried for 16hours. Dry dimethylformamide (200 ml) is added and the suspension heatedto 60° C. under a dry nitrogen atmosphere. Diiodomethane (1.61 ml, 20.00mmol) is added in two portions during 10 min. and the reaction mixtureleft for 4 days at 60° C. The solvent is removed under reduced pressure(0.05 mm Hg), before diethyl ether (140 ml), saturated aqueous sodiumhydrogen carbonate (50 ml) and water (50 ml) are added. The aqueouslayer is extracted with diethyl ether (6×60 ml) and the combined etherextracts washed with water (4×50 ml), dried (MgSO₄), and evaporated togive 1.06 g (34%) of the title compound. ¹H NMR (60 MHz, CDCl₃): δ5.81-6.61 (2×CH2=CH—, m), 5.84 (CH₂, s). This product may be used inaccordance with the invention, for example to crosslink acrylic acid andmethyl acrylate polymers.

Preparation 4 Chloromethyl (2-methacryloyloxy)ethyl carbonate

Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a solution ofchloromethyl chloroformate (0.89 ml, 11.00 mmol) and 2-hydroxyethylmethacrylate (1.22 ml, 10.00 mmol) in dichloromethane (12 ml) at 0° C.under a dry nitrogen atmosphere. After 21 hours at 20° C. the reactionmixture is washed with hydrochloric acid (1.00 M, 10 ml), saturatedaqueous sodium hydrogen carbonate (10 ml) and water (10 ml). The organicphase is dried (MgSO₄) and the solvent evaporated under reduced pressure(10 mm Hg) to give 1.97 g (88%) of the title compound. ¹H NMR (60 MHz,CDCl₃): δ 1.88 (CH₃, d, J=2 Hz), 4.35 (O—CH₂—CH₂—O, m), 5.47 (H—C═, m),5.63 (CH₂—Cl, s), 6.00 (H—C═, m)

Preparation 5 (2-Methacryloyloxy)ethyl methacryloyloxymethyl carbonate

A solution of potassium hydroxide (1.00 M, 5.00 ml) is added tomethacrylic acid (0.43 g, 5.00 mmol) at 0° C. and the solution freezedried during 16 hours. Dry dimethylformamide (50 ml) is added and to theresulting suspension is added chloromethyl (2-methacryloyloxy)ethylcarbonate (1.11 g, 5.00 mmol). 18-Crown-6 (0.066 g, 0.25 mmol) is addedas a catalyst and the reaction left under a dry nitrogen atmosphere.After 24 hours at 20° C. and 6 days at 4° C. the solvent is removedunder reduced pressure (0.05 mm Hg) and diethyl ether (30 ml) and water(20 ml) added. The aqueous layer is extracted with diethyl ether (3×20ml) and the combined ether extracts washed with water (20 ml), dried(MgSO₄) and evaporated to give 1.26 g (93%) of the title compound. ¹HNMR (60 MHz, CDCl₃): δ 1.97 (2×CH₃, m), 4.38 (O—CH₂—CH₂—O, m), 5.53(2×H—C═, m), 5.77 (CH₂, s), 6.07 (2×H—C═, m).

Preparation 6 Ethylene bis(chloromethyl carbonate)

Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a solution ofchloromethyl chloroformate (1.32 ml, 14.83 mmol) and ethylene glycol(0.28 ml, 5.00 mmol) in dichloromethane (10 ml) at 7° C. with goodstirring under a dry N₂ atmosphere. After 15 min. at 7° C. and 6 hoursat 20° C. the reaction mixture is transferred to a separating funnelwith the aid of dichloromethane (10 ml). The reaction mixture is washedwith hydrochloric acid (1.00 M, 10 ml), saturated aqueous sodiumhydrogen carbonate (10 ml) and water (10 ml). The organic phase is dried(MgSO₄) and the solvent evaporated under reduced pressure to give 1.12 g(90%) of the title product. ¹H NMR (300 MHz, CDCl₃): δ 4.48 (s,O—CH₂CH₂—O), 5.75 (s, 2×Cl—CH₂—O). ¹³C NMR (75 MHz, CDCl₃): δ 65.8(O—CH₂CH₂—O), 72.2 (2×Cl—CH₂—O), 153.0 (2×C═O)

Preparation 7 Bis(2-chloromethoxycarbonyloxyethyl)ether

Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a solution ofchloromethyl chloroformate (1.32 ml, 14.83 mmol) and diethylene glycol(0.47 ml, 5.00 mmol) in dichloromethane (10 ml) at 7° C. with goodstirring under a dry N₂ atmosphere. After 10 min. at 7° C. and 6 hoursat 20° C. the reaction mixture is transferred to a separating funnelwith the aid of dichloromethane (10 ml). The reaction mixture is washedwith hydrochloric acid (1.00 M, 10 ml), saturated aqueous sodiumhydrogen carbonate (10 ml) and water (10 ml). The organic phase is dried(MgSO₄) and the solvent evaporated under reduced pressure (10 mm Hg) togive 1.26 g (86%) title product. ¹H NMR (300 MHz, CDCl₃): δ 3.72 (m,2×CH₂—O), 4.34 (m, 2×CH₂—O—C═O), 5.71 (s, 2×Cl—CH₂—O). ¹³C NMR (75 MHz,CDCl₃): δ 67.6 (2×CH₂—C═), 68.5 (2×CH₂—O—C═O), 72.1 (2×Cl—CH₂—O), 153.2(2×C═O).

Preparation 8 1-Chloroethyl 2-methacryloyloxyethyl carbonate

Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a solution of1-chloroethyl chloroformate (1.20 ml, 11.00 mmol) and 2-hydroxyethylmethacrylate (1.22 ml, 10.00 mmol) in dichloromethane (12 ml) at 3° C.under a dry N₂ atmosphere. After 15 min. at 3° C. and 17 hours at 20° C.the reaction mixture is transferred to a separating funnel with the aidof dichloromethane (10 ml). The reaction mixture is washed withhydrochloric acid (1.00 M, 10 ml), saturated aqueous sodium hydrogencarbonate (10 ml) and water (2×10 ml). The organic phase is dried(MgSO₄) and the solvent evaporated under reduced pressure to give 1.76 g(74%) of the title product. ¹H NMR (60 MHz, CDCl₃): δ 1.85 (3 H, d, J=6Hz, CH ₃—CH), 1.96 (3 H,d, J=2 Hz, CH₃—C═), 5.55 (1 H, m, CH═), 6.10 (1H, m, CH═), 6.38 (1 H, k, J=6 Hz, CH—CH₃).

Preparation 9 Chloromethyl 4-acryloyloxybutyl carbonate

Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a solution ofchloromethyl chloroformate (0.98 ml, 11.00 mmol) and 4-hydroxybutylacrylate (1.38 ml, 10.00 mmol) in dichloromethane (12 ml) at 3° C. undera dry N₂ atmosphere. After 15 min. at 3° C. and 17 hours at 20° C. thereaction mixture is transferred to a separating funnel with the aid ofdichloromethane (10 ml). The reaction mixture is washed withhydrochloric acid (1.00 M, 10 ml), saturated aqueous sodium hydrogencarbonate (10 ml) and water (2×10 ml). The organic phase is dried(MgSO₄) and the solvent evaporated under reduced pressure to give 1.76 g(74%) of the title product. ¹H NMR (60 MHz, CDCl₃): δ 1.82 (4 H, m,CH₂—CH₂), 4.27 (4 H, m, 2×CH₂—O), 5.77 (2 H, s, Cl—CH₂—O), 5.8-6.7 (3 H,m, CH═CH₂).

Preparation 10 1-Chloroethyl 4-acryloyloxybutyl carbonate

Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a solution of1-chloroethyl chloroformate (1.20 ml, 11.00 mmol) and 4-hydroxybutylacrylate (1.38 ml, 10.00 mmol) in dichloromethane (12 ml) at 3° C. undera dry N₂ atmosphere. After 15 min. at 3° C. and 17 hours at 20° C. thereaction mixture is transferred to a separating funnel with the aid ofdichloromethane (10 ml). The reaction mixture is washed withhydrochloric acid (1.00 M, 10 ml), saturated aqueous sodium hydrogencarbonate (10 ml) and water (2×10 ml). The organic phase is dried(MgSO₄) and the solvent evaporated under reduced pressure to give 2.26 g(90%) of the title product. ¹H NMR (60 MHz, CDCl₃): δ 1.80 (4 H, m,CH₂—CH₂), 1.86 (3 H, d, J=5 Hz, CH₃), 4.24 (4 H, m, 2×CH₂—O), 5.7-6.6 (4H, m, CH═CH₂ and CH)

Preparation 11 1-Methacryloyloxyethyl 2-methacryloyloxyethyl carbonate

1-Chloroethyl 2-methacryloyloxyethyl carbonate (1.183 g, 5.00 mmol)prepared as described in Preparation 8 is added to a suspension offreeze dried potassium methacrylate (0.683 g, 5.50 mmol) and 18-crown-6(0.066 g, 0.25 mmol) in dimethylformamide (50 ml) under a dry N₂atmosphere. After 5 days at 20° C. the solvent is removed under reducedpressure and the residue dissolved by adding dichloromethane (60 ml) andwater (30 ml). After separating the phases the aqueous layer isextracted with dichloromethane (3×30 ml) and the combined organic phasewashed with saturated aqueous sodium hydrogen carbonate (50 ml). Theorganic phase is dried (MgSO₄) and the solvent removed under reducedpressure to give 1.10 g (77%) of the title product. ¹H NMR (60 MHz,CDCl₃): δ 1.63 (3 H, d, J=5 Hz, CH ₃—CH), 1.98 (6 H, s, 2×CH₃), 4.42 (4H, s, O—CH₂—CH₂—O), 5.62 (2 H, m, CH═), 6.15 (2 H, m, CH═), 6.84 (1 H,k, J=5 Hz, CH—CH₃).

Preparation 12 Acryloyloxymethyl 4-acryloyloxybutyl carbonate

Chloromethyl 4-acryloyloxybutyl carbonate (1.183 g, 5.00 mmol) preparedas described in Preparation 9 is added to a suspension of freeze driedpotassium acrylate (0.606 g, 5.50 mmol) and 18-crown-6 (0.066 g, 0.25mmol) in dimethylformamide (50 ml) under a dry N₂ atmosphere. After 5days at 20° C. the solvent is removed under reduced pressure and theresidue dissolved by adding dichloromethane (60 ml) and water (30 ml).After separating the phases the aqueous layer is extracted withdichloromethane (3×30 ml) and the combined organic phase washed withsaturated aqueous sodium hydrogen carbonate (50 ml). The organic phaseis dried (MgSO₄) and the solvent removed under reduced pressure to give1.24 g (91%) of the title product. ¹H NMR (60 MHz, CDCl₃): δ 1.82 (4 H,m, CH₂—CH₂), 4.23 (4 H, m, 2×CH₂—O), 5.88 (2 H, s, O—CH₂—O), 5.7-6.8 (6H, 2×CH═CH₂)

Preparation 13 1-Acryloyloxyethyl 4-acryloyloxybutyl carbonate

1-Chloroethyl 4-acryloyloxybutyl carbonate (1.253 g, 5.00 mmol) preparedas described in Preparation 10 is added to a suspension of freeze driedpotassium acrylate (0.606 g, 5.50 mmol) and 18-crown-6 (0.066 g, 0.25mmol) in dimethylformamide (50 ml) under a dry N₂ atmosphere. After 5days at 20° C. the solvent is removed under reduced pressure and theresidue dissolved by adding dichloromethane (60 ml) and water (30 ml).After separating the phases the aqueous layer is extracted withdichloromethane (3×30 ml) and the combined organic phase washed withsaturated aqueous sodium hydrogen carbonate (50 ml). The organic phaseis dried (MgSO₄) and the solvent removed under reduced pressure to give1.28 g (89%) of the title product. ¹H NMR (60 MHz, CDCl₃): δ 1.58 (3 H,d, J=5 Hz, CH ₃—CH), 1.80 (4 H, m, CH₂—CH₂), 4.24 (4 H, m, 2×CH₂—O),5.7-6.7 (6 H, m, 2×CH═CH₂), 6.87 (1 H, k, J=5 Hz, CH—CH₃).

Preparation 14 a) Methylene bis(3,3-dimethoxypropionate)

Cesium 3,3-dimethoxypropionate (19.95 g, 75 mmol) is added to dry DMF(1000 ml). Diiodomethane (10.04 g, 37.5 mmol) is added to the suspensionand the reaction mixture is stirred for 2 days at 60° C. under a dry N₂atmosphere. DMF is removed under reduced pressure (0.01 mmHg). Diethylether (500 ml) is added to the residue, which is then washed withsaturated aqueous sodium hydrogen carbonate (250 ml). The aqueous layeris extracted with diethyl ether (5×75 ml). The combined ether extractsare washed with water (2×100 ml), dried (MgSO₄) and evaporated to give7.1 g (72%) product. ¹H NMR (300 MHz, CDCl₃): δ 2.61 (CH₂, d), 3.26(CH₃, s).

b) Methylene bis(3-methoxypropenoate)

Methylene bis(3,3-dimethoxypropionate) (14.01 g, 50 mmol) prepared asdescribed in (a) above and a catalytic amount of p-toluene sulfonic acidis added to toluene (250 ml). The methanol is removed by warming thereaction under an N₂ atmosphere. When the reaction is complete thetoluene is distilled off under reduced pressure. Diethyl ether (250 ml)is added and the mixture is washed with saturated aqueous sodiumhydrogen carbonate (5×50 ml) and water (3×50 ml). The organic layer isdried (MgSO₄) before evaporation to give 8.52 g (79%) product. ¹H NMR(300 MHz, CDCl₃): δ 3.65 (2×CH₃, s), 5.2 (2×CH, d), 5.8 (O—CH₂—O), 7.65(2×CH₂, d).

Preparation 15 a) Methylene bis(10-undecenoate)

10-Undecylenic acid (12.75 g, 75 mmol) is dissolved in 100 ml water.Cesium carbonate (13.04 g, 40 mmol) is added to the mixture. The wateris removed under reduced pressure and the salt dried for 2 hours invacuo. The cesium salt is mixed with 150 ml DMF and diiodomethane isadded to the solution. The reaction is stirred for 3 days at 60° C.under an N₂ atmosphere. DMF is then removed under reduced pressure. Theresidue is purified through silica gel with hexane/ ethyl acetate (8:2)as eluant. The solvent is evaporated to give 7.18 g (54%) product. ¹HNMR (300 MHz, CDCl₃): δ 1.2-1.4 (10×CH₂, m), 1.6 (2×CH₂, m), 2.0 (2×CH₂,m), 2.19 (2×CH₂, t), 4.9 (2×H₂C═, m), 5.88 (O—CH₂—O, s), 5.9 (2×HC═, m).¹³C NMR (300 MHz, CDCl₃): δ 24.92-33.98 (8×CH₂), 79.04 (O—CH₂—O), 114.18(═CH₂), 139.11 (═CH), 172.48 (C═O).

b) Methylene bis(10-epoxyundecanoate)

Methylene bis(10-undecenoate) (8.8 g, 25 mmol) prepared as described in(a) above is added under an N₂ atmosphere to methylene chloride andcooled to 0° C. Metachloroperbenzoic acid 55% (15.75 g, 50 mmol) isadded to methylene chloride (150 ml) and the organic layer is separatedand dried (MgSO₄). The metachloroperbenzoic acid is then added dropwiseto the diester. After completed addition the temperature is increased to25° C. After 5 hours the reaction is complete. The mixture is washedwith saturated aqueous sodium sulphite (75 ml) and saturated aqueoussodium hydrogen carbonate (2×75 ml). The organic layer is purified onneutral aluminium oxide. The solvent is removed under reduced pressureto yield 8.45 g (82%) product. ¹H NMR (300 MHz, CDCl₃): δ 1.2-1.7(14×CH₂, m), 2.35 (2×CH₂CO, t), 2.45 (2×CH,q), 2.75 (2×CH,q), 2.90(2×CH,m), 5.75 (O—CH₂—O). ¹³C NMR (300 MHz, CDCl₃): δ 24.58 (CH₂), 25.99(CH₂), 28.94 (CH₂), 29.09 (CH₂), 29.32 (2×CH₂), 32.45 (CH₂), 33.92(CH₂), 47.06 (CH₂—O), 52.36 (CH—O), 79.06 (O—CH₂—O), 172.2 (C═O).

Preparation 16 Methylene bis(4-epoxypentanoate)

Metachloroperbenzoic acid (15.68 g, 55%, 50 mmol) is dissolved inmethylene chloride (200 ml). Water is separated and the organic layer isdried (MgSO₄). The resulting metachloroperbenzoic acid solution is addeddropwise to methylene bis(4-pentenoate) (4.10 g, 19 mmol) dissolved inmethylene chloride (50 ml). The mixture is stirred at ambienttemperature under nitrogen for 12 hrs, whereafter the reaction mixtureis washed with saturated aqueous sodium bicarbonate solution (50 ml),water (50 ml), dried (MgSO₄) and evaporated to give 3.61 g (78%) of thetitle compound as a crystalline product. ¹H NMR (300 MHz, CDCl₃): δ1.70-1.85 (2×CH,m), 1.95-2.10 (2×CH,m), 2.50-2.55 (2×CH, 2×CH₂,m), 2.75(2×CH,t), 3.0 (2×CH,m), 5.8 (O—CH₂—O, s). ¹³C NMR (75 MHz, CDCl₃): δ 27(2×CH₂), 30 (2×CH₂), 47 (2×CH₂), 51 (2×CH), 79.8 (O—CH₂—O), 171.8(2×C═O)

Preparation 17 Methylene bis(2-butenoate)

Vinylacetic acid (4.3 g, 50 mmol) is added to an aqueous cesiumcarbonate solution (50 ml). The mixture is stirred for 5 min. and thenevaporated, and the residue is dried under vacuum for 2 hrs. Theresulting cesium salt and diiodomethane are added to dimethylformamide(200 ml) and the mixture is stirred for 24 hrs. at 50° C. undernitrogen, whereafter the dimethylformamide is removed under reducedpressure. The residue is dissolved in diethyl ether (100 ml) and washedwith saturated aqueous sodium bicarbonate (25 ml) and water (25 ml). Theorganic layer is dried (MgSO₄) and evaporated to give 1.32 g (29%)product. ¹H NMR (300 MHz, CDCl₃): δ 1.9 (2×H₂,m), 5.8-5.9 (2×CH,m), 5.9(OCH₂O, s), 7.0-7.1 (2×CH,m)

Preparation 18 Methylene bis(chloroacetate)

Chloroacetic anhydride (12.75 g, 75 mmol), paraformaldehyde (2.25 g, 75mmol) and conc. sulfuric acid (15 drops) are added to methylene chloride(15 ml). The mixture is stirred for 24 hrs. at 50° C. under nitrogen,whereafter the reaction mixture is extracted with saturated aqueouspotassium carbonate until carbon dioxide emission ends. The organiclayer is dried (MgSO₄), evaporated to dryness and the residue isdistilled (80° C., 0.15 mmHg) to yield 10.2 g (57%) product. ¹H NMR (200MHz, CDCl₃): δ 4.1 (2×CH₂Cl,s), 5.9 (CH₂,s). ¹³C NMR (200 MHz, CDCl₃): δ41.1 (CH₂Cl), 81.4 (O—CH₂—O), 166.4 (CO)

Preparation 19 Methylene bis(4-oxopentanoate)

4-Oxopentanoic acid (11.6 g, 100 mmol) is dissolved in acetonitrile (70ml), and 1,8-diazabicyclo[5.4.0]undec-7-ene (15.25 g, 100 mmol) dilutedwith acetonitrile (30 ml) is added. Diiodomethane (13.4 g, 50 mmol) isadded in one batch, and the reaction mixture is refluxed under anitrogen atmosphere. After 2 hours, gas chromatography indicates fullconsumption of diiodomethane. The solvent is removed in vacuo and theresidual brown oil is transferred to a separation funnel with ethylacetate (200 ml) and water (75 ml). The organic phase is washed with 1Msodium bicarbonate (25 ml) and water (3×25 ml), dried over MgSO₄, andthe solvent is removed in vacuo to yield the title compound (10 g). ¹HNMR: δ 2.19 (2×CH₂, s), 2.760-2.804 (2×CH₂, t), 2.600-2.645 (2×CH₂, t),5.735 (CH₂ bridge, s)

Preparation 20 Methylene bis(succinimidylazelate)

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.49 g,7.71 mmol) was added in portions to a stirred solution of methylenebis(hydrogen azelate) from Example 25 (1.00 g, 2.57 mmol) andN-hydroxysuccinimide (0.89 g, 7.71 mmol) in dry dimethylformamide atambient temperature. After 20 hours stirring, the reaction mixture waspoured into ice-water and the product precipitated as an oil. Thecolourless oil was dissolved in diethylether (50 ml), washed with water(3×10 ml) and dried over MgSO₄. The solvent was removed under reducedpressure and hexane (5 ml) was added to the oily product. After sevendays storage at 4° C. the oil had crystallized to a white, waxy solid.Yield: 1.50 g (69%). m.p.: 45-47° C. ¹³C NMR (75 MHz, CDCl₃) δ: 24.42,24.46, 25.59, 28.48, 28.63, 30.85, 33.82, 79.61, 168.6, 169.30, 172.34.

Preparation 21 Methylene bis(sulphosuccinimidylazelate) sodium salt

Methylene bis(hydrogen azelate) (0.38 g, 1 mmol), N-hydroxysuccinimidesodium salt (0.48 g, 2.2 mmol) and dicyclohexylcarbodiimide (0.45 g,2.2. mmol) were dissolved in dimethylformamide (10 ml). The suspensionwas stirred overnight at room temperature under an atmosphere ofnitrogen. The reaction mixture was filtered and purified by reversedphase chromatography (RP-8) with water/acetonitrile (1:1) as eluant togive the title compound.

Preparation 22 a) Methylene bis(10,11-dihydroxyundecanoate)

N-Methylmorpholine-N-oxide (13.5 g, 11 mmol) and methylenebis(10-undecenoate) from Preparation 15(b) (19 g, 5 mmol) were dissolvedin 400 ml of a mixture of tetrahydrofuran and water (3:1 v/v). Acatalytic amount of osmium tetroxide was added, and the solution stirredat ambient temperature for 20 hours. TLC indicated complete consumptionof the starting material. Excess sodium hydrogen sulphite and sodiumchloride were then added to the reaction mixture. The product wasextracted from the resulting mixture with ethyl acetate (400 ml) and thewater phase was washed with ethyl acetate (3×50 ml). The combinedorganic phases were dried and evaporated, and the product recrystallisedfrom tetrahydrofuran to yield 14.5 g (68%) of the product as a whitesolid. ¹³C NMR (45 MHz) CD₃OD: δ 24.6-34.0 (16×CH₂), 66.6 (2×CH₂OH),72.3 (2×CHOH), 79.2 (O—CH₂—O), 174.0 (2×C═O)

b) Methylene bis(10-oxodecanoate)

Methylene bis(10,11-dihydroxyundecanoate) (2.24 g, 5 mmol) was dissolvedin 150 ml tetrahydrfuran. Sodium metaperiodate (2.06 g, 10 mmol) wasdissolved in 150 ml water and added dropwise to the tetrahydrofuransolution. TLC indicated full consumption of the diol after 60 minutes,whereupon sodium chloride was added to the reaction mixture until thetwo phases separated. The water phase was extracted with diethyl ether(3×50 ml). The combined organic phases was dried with magnesium sulphateand evaporated to give the title product as an oil, 1.43 g (74%). ¹³CNMR (45 MHz) CDCl₃: δ 21.9-43.9 (16×CH₂), 79.1 (O—CH₂—O), 173.0 (2×C═O),202.6 (2×CHO).

EXAMPLE 1

1. Gas-filled albumin microspheres are prepared according to EP-A-0359246 and resuspended to homogeneity by gentle rolling on a vial roller.

2. 25 ml of the suspension are poured into a 25 ml separating funnel andleft for 30 min. The bottom 20 ml are discarded.

3. To the remaining 5 ml is added 20 ml of a phosphate buffer (20 mMNaPO₄, pH 7.0), and the resulting suspension is transferred to a vialwith a cap septum.

4. The vial is centrifuged upside down at 170×g for 5 min.

5. The solution underneath the microsphere layer is withdrawn using asyringe, and the microspheres are resuspended in 25 ml of the phosphatebuffer by 10 min of gentle rolling.

6. Points 4 and 5 are repeated twice.

7. The resulting suspension is centrifuged as in point 4, and themicrospheres are resuspended in the phosphate buffer to a finalconcentration of about 5×10⁸ particles per ml.

8. The crosslinker methylene bis(α-formylacetate), prepared as describedin Preparation 1, is added to the suspension, and the crosslinkingreaction is allowed to proceed for the desired time (usually 30-60 min)under gentle rolling.

9. 1.5 M Tris-HCl-buffer (pH 8.8) is added to a final concentration of0.25 M, and the suspension is rolled gently for 10 min.

10. The vial is centrifuged as in point 4, and the solution underneaththe microsphere layer is removed as in point 5.

11. The microspheres are resuspended in phosphate buffer (same volume asfinal volume in point 9), and the suspension is rolled for 10 min.

12. Points 10 and 11 are repeated twice.

13. The resulting suspension is centrifuged as in point 4, and themicrospheres are resuspended in the phosphate buffer to a finalconcentration of about 5×10⁸ particles per ml.

14. This final suspension of crosslinked gas/albumin microspheres isstored at 4° C.

EXAMPLE 2-22

The procedure of Example 1 is repeated using crosslinking agentsprepared as described in Preparations 2-22, except that dimethylsuplhoxide is used in place of phosphate buffer in the processing of thegas-filled albumin microspheres according to steps 3-7 and thecrosslinking agent is added in step 8 as a solution in dimethylsulphoxide.

The number and size distribution of the products are determined byCoulter counter analysis.

1. Contrast agents for use in diagnostic ultrasound studies comprisingmicrobubbles of gas or a gas precursor encapsulated in a protein shellcharacterised in that the said protein is crosslinked with crosslinkinggroupings containing biodegradable linkages.
 2. Contrast agents asclaimed in claim 1 wherein the crosslinking groupings containbiodegradable linkages selected from amide, imide, imine, ester,anhydride, acetal, carbamate, carbonate, carbonate ester and disulphidegroups.
 3. Contrast agents as claimed in claim 2 wherein thecrosslinking groups contain biodegradable linkages of formula—(Y)_(n)—CO—O—C(R¹R²)—O—CO—(Z)_(n)— (where Y and Z, which may be thesame or different, are —O—, —S— or —NR³—; R¹ and R², which may be thesame or different, are hydrogen atoms or carbon-attached monovalentorganic groups or together represent a carbon-attached divalent organicgroup; R³ is a hydrogen atom or an organic group; and the symbols n,which may be the same or different, are zero or 1).
 4. Contrast agentsas claimed in any of the preceding claims wherein the protein isalbumin, gelatin or globulin.
 5. Contrast agents as claimed in claim 4wherein the protein is human serum albumin.
 6. Contrast agents asclaimed in any of the preceding claims further containing an inorganicparticulate stabiliser.
 7. A process for the preparation of a contrastagent as claimed in claim 1 which comprises encapsulating a gas or gasprecursor in a protein and crosslinking the protein with crosslinkinggroups containing biodegradable linkages before, during or after saidencapsulation.
 8. A process as claimed in claim 7 wherein crosslinkingis effected after encapsulation.
 9. A process as claimed in claim 7 orclaim 8 wherein crosslinking is effected using a crosslinking agent offormula (I)A¹-X-A²   (I) (where X is a linking group containing one or morebiodegradable linkages as defined in claim 2 or claim 3 and A¹ and A²,which may be the same or different, are functional groups reactive withproteins).
 10. A process as claimed in claim 9 in which A¹ and A² areboth aldehyde groups.
 11. A process as claimed in any of claims 8 to 10wherein encapsulation is effected by agitation or sonication of theprotein in an aqueous medium to yield a protein foam which is dried andthereafter suspended in a solution of the crosslinking agent in a polarorganic solvent
 12. A process as claimed in claim 11 in which thecrosslinking agent is a compound of formula (I) as defined in claim 9 inwhich A¹ and A² are both O-linked sulphonated N-hydroxysuccinimidylresidues.